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Rose Et Al 1978. Poliovirus Shut Off Celular
Rose Et Al 1978. Poliovirus Shut Off Celular
USA
Vol. 75, No. 6, pp. 2732-2736, June 1978
Biochemistry
Abbreviations: VSV, vesicular stomatitis virus; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; elF, eukaryotic initiation
factor.
2732
(17).
Ohr
I hr
2hr
3hr
i.-i_ -2
Go b|
2733
"
11
801
-1~~~~~~~~~~A
....
"7_-
U)
O ..e
NS-
4)
60
-Sc
0)
Z
e
*ON
M-
f aferinfecton
FIG. 1. Kinetics of poliovirus-induced inhibition of translation. (A) Inhibition in vivo. HeLa cells were infected with poliovirus and pulse
labeled with [(3S]methionine (10 #Ci/ml). Cytoplasmic proteins were subjected to gel electrophoresis (10% acrylamide) and detected by autoradiography. Times given above lanes 1-5 refer to times after virus adsorption at which each 15-min pulse label was begun. The 0 time sample
was from cells that were mock-infected. Equal radioactivity was placed into each lane. Total acid-precipitable incorporation (cpm per 0.01 ml
of cells) was 1.7 X 104 (0 time), 4.4 X 104 (1 hr), 5.5 X 104 (2 hr), 6.6 X 104 (2.5 hi), and 7.6 X 104 (3 hr). (B) Inhibition in vitro. [35S]Methioninelabeled proteins synthesized in vitro were subjected to gel electrophoresis (12.5% acrylamide) and detected by fluorography (22). Times above
lanes 1-10 indicate time after virus adsorption at which the cells were harvested for preparation of the three translation systems. The 0 time
extract was from cells that were mock-infected. Ten microliters of each translation reaction were analyzed: lanes 1, 4,6, and 8, no added RNA;
lanes 2,5,7, and 9, VSV mRNA; lanes 3 and 10, poliovirus mRNA. Lane 11 shows marker proteins synthesized in vivo 3 hr after poliovirus infection.
Positions of NCVP1A and NCVP2 as well as unlabeled VSV virion proteins are indicated. (C) Quantitation of inhibition in vitro. Regions of
the gel (B) containing proteins synthesized in vitro were excised and radioactivity was determined in a scintillation counter. Background radioactivity from the same regions (no added RNA) was subtracted. Radioactivity in N and NS proteins (-) and M protein (X) as well as total
poliovirus proteins (a) is plotted as percent of synthesis obtained in the mock-infected extract.
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Ea
0.
'al0U
i.
a
CD
min at 500
FIG. 3. Purification and heat inactivation of eIF-4B. (A) Phosphocellulose chromatography. Fractions obtained during gradient
elution of eIF-4B from phosphoceflulose (step 5, ref. 19) were pooled,
concentrated, and assayed for eIF-4B activity in the initiation factor-dependent system b (19) and for stimulation of VSV M protein
and N + NS protein synthesis in an infected lysate as in Figs. 1C and
2. Pools 1-4 eluted at KC1 concentrations of 0.1 M, 0.13-0.23 M,
0.23-0.33 M, and 0.33-4 M, respectively. 0, eIF-4B activity-, 0,
stimulation of N plus NS synthesis; X, stimulation of M synthesis.
(B) Heat inactivation. Samples of step 4 eIF-4B in 20 mM Tris-HC1,
pH 7.6/100 mM KCl/0.1 mM EDTA/14 mM 2-mercaptoethanoV10%
glycerol were incubated at 50 for 1, 2, 5, and 10 min, and assayed as
described in A.
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Table 1. Effects of eIF-4B and eIF-2 on VSV and poliovirus mRNA translation
Infected extract
Uninfected extract
eIF-2
eIF-2
eIF-4B eIF-4B
+RNA (prep. 1)* (prep. 2)* eIF-2*
1,440
2,300
2,310
19,100
725
959
1,330
11,000
32,500
10,700
38,600
101,000
ND
ND
ND
2,230
eIF-4B
(prep. 2)* -RNA
158
29,000
67
14,900
eIF-4B eIF-4B
+RNA (prep. 1)* (prep. 2)* eIF-2*
155
418
369
1,100
53
186
66
1,150
eIF-4B
(prep. 2)*
4,250
2,770
89,500 1,541 24,400
7,210
25,800 58,100 51,000
ND
62
243
ND
861
ND
ND
Translation of VSV mRNAs, poliovirus mRNA, and rabbit globin mRNA was as described in Materials and Methods. The amounts of initiation
factors added per 25-;l reaction mixture were approximately 1.5 ug (eIF-4B, prep. 1), 0.15 Mg (eIF-4B, prep. 2), and 0.5 Mg (eIF-2, purified through
step 3, ref. 19). The proteins synthesized in response to each mRNA were analyzed by gel electrophoresis, and protein bands were quantitated
as described in the legend of Fig. 1C. The numbers given are the cpm in the protein bands or in the equivalent region of the gel for translation
without RNA. The stimulatory effect of eIF-2 on VSV mRNA translation was variable (4- to 15-fold) in different extracts. ND = not determined.
* In the
presence of RNA.
Protein
N + NS
M
Polio
Globin
-RNA
208
63
1,206
79
stimulation of VSV protein synthesis in preliminary experiments, we also tested this factor alone and in combination with
eIF-4B (prep. 2) in this experiment (Table 1). Addition of eIF-2
resulted in greater than 10-fold stimulation of VSV mRNA
translation in uninfected lysates in the presence or absence of
eIF-4B. A similar 10-fold increase in VSV translation could be
seen in the infected lysate when eIF-2 was assayed in the
presence of eIF-4B (prep. 2). Poliovirus mRNA translation was
also stimulated by eIF-2 (about 3-fold) in lysates from infected
or uninfected cells. These results indicate that eIF-2 can be
limiting for translation in both the infected and uninfected
lysates but does not appear to be directly involved in the inhibition of translation by poliovirus.
In a separate experiment, translation of globin mRNA in
infected and uninfected cell extracts was assayed. The results
were qualitatively similar to those obtained with VSV mRNA;
some globin synthesis, however, was clearly detectable in the
infected lysate. This suggests that all mRNAs may not show the
same extent of translation inhibition in infected lysates.
Inactivation of eIF-4B In Vitro. To examine the possibility
that inactivation of eIF-4B might occur in vitro, we measured
translation of VSV mRNA in mixed extracts from poliovirusinfected and uninfected cells. The results (Fig. 4) show that VSV
mRNA translation occurred in extracts from uninfected and
poliovirus-infected cells that had been mixed prior to translation. The level of translation was approximately proportional
to the amount of uninfected extract present in the mixture. This
result shows that there was not an excess -of an inhibitor in the
infected lysate that could cause a rapid inactivation of translation. If the mixed extracts were preincubated for 30 or 60 min
prior to translation, a loss of ability to translate VSV mRNA was
seen (Fig. 4). That this in vitro inactivation was due to loss of
eIF-4B activity was shown by addition of eIF-4B after a 30-min
preincubation of the mixed extract. This addition increased
VSV mRNA translation to approximately the unincubated level
(Fig. 4). Control experiments showed that the extract from
uninfected cells lost only 10% of its activity after 60 min of
preincubation (Fig. 4).
DISCUSSION
We have described here an in vitro translation system from
poliovirus-infected cells that reproduces the selective translation
inhibition of host and VSV mRNA translation seen in vivo. This
inhibition can be overcome by addition of a single purified
translation initiation factor, eIF-4B. Also, lysates from poliovirus-infected cells can inactivate eIF-4B when it is added to
the lysate (data not shown) or in mixed lysates from infected
(25).
Because poliovirus mRNA translation occurs normally in
lysates from infected cells and is not stimulated by added
u
.0
100
60
~~60 ~
40
40~~~
2 20
30
60
Preincubation, min
FIG. 4. Translation in mixed lysates. In vitro translation was in
uninfected lysates or in poliovirus-infected and uninfected lysates
mixed at the indicated ratios. Lysates were preincubated for the indicated times prior to addition of translation reaction components
and VSV mRNA. VSV M protein synthesis was quantitated as in the
legend of Fig. 1C. The synthesis of M protein in the mixture of infected and uninfected lysates (1:1, 30-min preincubation) with eIF-4B
(prep. 1, approximately 1.5 Mg) added after preincubation is indicated,
*. X, Uninfected, 551 cpm at 100%; 0, uninfected + infected (4:1),
443 cpm at 100%; 0, uninfected + infected (1:1), 225 cpm at 100%.
2736